The present invention relates to a process for preparing a water-insoluble immobilized enzyme composition.
Methods for immobilizing enzymes are well known in the art. The terms "immobilizing enzymes" and "immobilized enzymes" as applied in the present specification mean that these enzymes are rendered essentially insoluble, so that they are rendered reusable and can be utilized in continuous processes. For this purpose enzymes are attached to insoluble support materials by means of adsorption or covalent bonding. Conventionally the support material is loaded with the enzyme by treating it with an enzyme solution.
Organic carrier materials (such as cellulose, nylon, or polyacrylamide) exhibit serious disadvantages since they do not possess sufficient mechanical strength, can be affected by the action of solvents, and are sensitive to changes in the pH-value or the ion concentration in the surrounding medium. Furthermore, several of these organic materials are susceptible to being attacked by microbes which may cause a loosening of the bond between the enzyme and the support material.
Therefore, inorganic materials have been suggested as carrier materials onto which enzymes may be attached by means of adsorption or covalent bonding. Which type of attachment is preferred depends on the properties of the substrate for which, and on the conditions under which the enzyme is to be utilized. If the substrate is in a medium which contains a high salt concentration, an attachment by mere adsorption cannot be used, since desorption of the adsorbed enzyme molecules may occur. Therefore, a covalent bonding between the enzyme and the carrier is preferred. For this, the carrier surface must include a sufficient amount of functional groups which are capable of forming a covalent bond with the respective enzyme. Since most inorganic carrier materials do not contain such specific functional groups, a pretreatment of the carrier surface is necessary. A conventional method for providing functional organic groups in the surface of an inorganic carrier material comprises loading the inorganic materials with silanes, which attach to the surface of the carrier material and provide the latter with functional organic groups, preferably alkylamino groups, which are capable of forming a covalent bond with organic compounds. Treatment of the inorganic carrier material with glutardialdehyde, sulfuryl chloride, thionyl chloride, or cyanur chloride, has also been tried.
It also is possible to provide the surface of the inorganic carrier material with a coating of a water-insoluble organic polymer which comprises free functional groups, for example, a polyacrolein which comprises between about 10 and about 70% of free aldehyde groups relative to the number of monomer units.
Aluminum oxides, nickel oxide, iron oxide, titanium oxide, zirconium oxide, hydroxyapatite, silicates, and porous glass have been proposed as porous inorganic carrier materials. The pore-structure within these carrier materials has to be such that the enzyme is able to reach the inner surface of the support particles. Yet, with regard to which additional properties of the carrier material are desirable, i.e., which are the optimum pore-size distributions and/or surface areas, the available information differs largely from each other.
Independently of the type of bonding between enzyme and support material, and the use of any of the aforementioned support materials, it has not yet been possible to immobilize enzymes in such a manner that the specific activity of the enzyme in the immobilized state reaches the value of the specific activity of the enzyme in the free state. According to D. L. Latigue (see Immobilized Enzymes for Industrial Reactors, London, 1975, p. 127), even under the most favorable immobilizing conditions only 80% at the most of the enzyme which is attached to the support material is present in active form.